The advent of effective anti-viral therapies including anti viral pharmaceuticals, immune based therapies and vaccines has created a market for therapeutic susceptibility testing much like applying antibiotic disks to agar plates to test bacterial susceptibilities. Traditional patient monitoring tools such as CD4 T cell counts and viral load evaluations provide little information on the drug susceptibility patterns of HIV and other viruses. Resistance testing, specifically phenotypic testing, overcomes this barrier by helping clinicians understand exactly which drugs may or may not work for a particular patient. With this information, physicians can optimize treatment regimens for improved patient outcomes. Currently, there are two methods of drug resistance testing:
Genotyping
Genotypic resistance means that a change has occurred in the specific sequencing of nucleotides that comprise a codon. A change in a condon will lead to amino acid and structural changes in the protein, resulting in a viral mutation that can be less susceptible to antiviral medications. During genotypic analysis, the RNA or DNA fragments found in solution that correspond to genes coding for the reverse transcriptase and protease enzymes are evaluated to determine if genetic mutations are present. The results are compared to preestablished mutation patters that have shown resistance to specific antiviral drugs. If the genetic mutations present in a patient sample match the preestablished resistance mutations for a certain drug, then the virus is assumed to be resistant to that drug. Genotypic analysis is an indirect measure of drug susceptibility that offers a prediction of resistance. The results are based on averages and cannot be directly linked to treatment response.
Phenotyping
Phenotypic analysis is a direct, quantitative measure of drug resistance that does not require interpretation of complex genetic mutation patterns. Rather, phenotypic testing measures the ability of a specific viral strain to grow in vitro in the presence of a drug inhibitor. Drug susceptibility is defined by the amount of drug required to inhibit the patient virus by 50% (IC50) or 90% (IC90) as compared to a drug-sensitive reference virus. The patient virus is less susceptible to a particular drug when more of the drug is required to inhibit viral activity, versus the amount of drug required to inhibit the reference virus.
Because the virus is directly exposed to each of the available antiretroviral medications, results can be directly linked to treatment response. For example, if the patient virus shows resistance to a particular drug, that drug can be avoided or omitted from the patient""s treatment regimen, allowing the physician to design a treatment plan that is more likely to be effective for a longer period of time.
Currently available genotyping assays require gene sequencing to detect resistance mutations. Currently available phenotyping assay require cloning of the patients isolate into either specialized vectors (Virologic) or cells (Virco) and subsequent exposure to the drugs to be tested. Both genotyping and phenotyping can take up to two (2) weeks for the results because of the labor and analysis required (genotyping) or long culture times because of the insensitivity of the detection system (p24 antigen). Further, all genotyping and phenotyping assays assume that resistance is inherent in the virus, in particular, in the gene sequence of the virus.
The present invention provides a technique that addresses the fact that viral resistance may be a function of the cell types infected and the point at which the antiviral compound inhibits viral replication in the lifecycle of the virus within these cell types. The present invention can detect if viral resistance is due to the fact that viruses may not proceed through a normal lifecycle in certain cells, such as monocytes or dendritic cells. The present invention does this by mixing virally infected subject cells with tagged target cells having a marker. Viral production is then stimulated and the mixture is subjected to an antiviral compound. The mixture is then analyzed for viral production in the target cells.